Mesenchymal Stem Cells as a Feeder Layer Can Prevent Apoptosis of Expanded Hematopoietic Stem Cells Derived from Cord Blood

Umbilical cord blood (UCB) has been used for transplantation in the treatment of hematologic disorders as a source of hematopoietic stem cells (HSCs). Because of insufficient number of cord blood CD34+ cells, the expansion of these cells seems to be important for clinical application. Mesenchymal stromal cells (MSCs), playing an important role in HSCs maintenance, were used as feeder layer. Apoptosis and cell cycle distribution of expanded cells were analyzed in MSCs co-culture and cytokine conditions and results were compared. Three culture conditions of cord blood HSCs were prepared ex-vivo for 14 days: cytokines (SCF, TPO and Flt3L) with MSCs feeder layer, cytokines without MSCs feeder layer and co-culture with MSCs without cytokines. Expansion was followed by measuring the total nucleated cells (TNCs), CD34+‏ cells and colony-forming unit (CFU) output. Flow cytometry analysis of stained cells by annexin V and propidium iodide was performed for detection of apoptosis rate and cell cycle distribution in expanded cells. Maximum cord blood CD34+ cells expansion was observed in day 10. The mean fold change of TNCs and CD34+ cells at day 10 in the co-culture system with cytokines was significantly higher than the cytokine culture without MSCs feeder layer and co-culture system without cytokines (n=6, p=0.023). The highest apoptosis rate and the least number of cells in Go/G1 phase were observed in cytokine culture without feeder layer (p=0.041). The expansion of cord blood HSCs on MSCs as a feeder layer resulted in higher proliferation and reduction in apoptosis rate.

Physiologically, stromal layer cells produce soluble factors which maintain the primitive characteristics of HSCs and the direct interaction between the HSC and MSC can catalyze this process (9). In this study we evaluated the rate of HSCs proliferation and apoptosis in the presence of mesenchymal stromal cells, Flt3-L, SCF and TPO.

Statistical Analysis
Results obtained from multiple experiments are expressed as the mean ± standard deviation (SD).
The data were analyzed using the t-test. Probability values<0.05 defined significant differences between test points.

MSCs Immunophenotyping
Isolated bone marrow MSCs were characterized by flow cytometric analysis of specific surface antigens. MSCs were found to be positive for the following adhesion molecules: CD44, CD166 and CD105, CD90 which together were considered as markers for MSCs. The MSCs were negative for haematopoietic lineage markers, CD34 and CD45.

Osteogenic differentiation assay
Potential osteogenic differentiation of bone marrow derived MSCs was assayed by alizarin red staining and evaluation of alkaline phosphatase activity. Both alizarin red and alkaline phosphatase activity showed a positive reaction ( Figure 1).

Ex-vivo expansion of CD34+ enriched cells
The purity of separated CD34 + cells determined by flow cytometry analysis, was 88% ± 12.
Moreover, 31.45% ± 7 of them were positive for CD38 marker. Lineage specific CD markers expression was low (n=3) (Figure 2). in the co-culture system with cytokines (n=6) (Figure 4). However, in co-culture system without cytokine, TNC and CD34+ cell numbers were increased up to 4.88 ± 1.5 and 2 folds respectively.

Ex-vivo expansion of human cord blood
Expansion of CD34 + selected cord blood cells at 14 th day of both cytokine culture and coculture with cytokines was decreased and a relative differentiation was also observed. The mean fold change of TNC and CD34+ cells at 10 th day in the co-culture system with cytokines was significantly higher than the cytokine culture without MSCs feeder layer and co-culture system without cytokines (n=6, p=0.023). However, in the co-culture system without cytokine, TNC and CD34 + cell numbers increased up to 2 folds and cell viability remained 90% after 14 days.

Colony-forming cell assays
The highest CFU fold change was also observed in cytokine culture with MSCs at 10 th day (90 ± 24). The CFUs increase in cytokine culture without MSCs at 10 th day was 87 ± 13, and in coculture with MSC was 52 ± 9. So the mean fold change of CFU at 10 th day in both cytokine cultures with and without MSCs feeder layer were significantly higher than the co-culture system without cytokines (n=3, p=0.037), but there was no significant differences between the two cytokine cultures with and without MSCs feeder layer (Figures 4, 5).

Cell Cycle Distribution Analysis
The distribution of expanded cells in percentage was 56.5 ± 1 at G0/G1, 9.46 ± 1. Our results showed that the population of cells in Go/G1 phase in two co-culture condition was significantly higher than cytokine culture and in coculture without cytokine was also higher than coculture with cytokine (p=0.041). The population of cells in G2/M phase in co-culture with cytokine was higher than other conditions (p=0.034) But there were no significant differences between the population of cells in S phase ( Figure 6).

Apoptosis analysis
Apoptosis analysis was performed by Annexin V and PI staining on expanded cells cultured in all three culture conditions at day 10 th of culture. The percentage of apoptotic cells was 8 ± 1.2% PI + , 6.86 ± 0.3% Annexin + in co-culture without cytokine, 17.18 ± 0.7% PI + , 14.94 ± 1% Annexin + in coculture with cytokine and 35.77 ± 4.07% PI + , 27.03   showed that the apoptosis rate in cytokine culture was significantly higher than the two co-cultures condition (P<0.005). Apoptosis rate in co-culture with cytokine was higher than co-culture without cytokine (P<0.005) (Figures 7, 8).  Another way is expansion of HSCs in co-culture with mesenchymal stem cells that produce a more "natural" haematopoietic microenvironment for proliferation, self renewal and differentiation of HSCs (11). Prevention of apoptosis is an important strategy to improve stem cell engraftment in preclinical and clinical settings (12). In this study, MSCs have been demonstrated to serve as a feeder layer and to maintain HSCs in an undifferentiated state (16). The rate of expansion in culture conditions containing cytokine decreased at 14 th day of expansion, but in the co-culture system without cytokine 4 fold expansion was observed constantly during 14 days. Song et al. (2010) claimed that co-cultures of hematopoietic cells with bone marrow derived mesenchymal stem cells without added cytokines, were able to preserve repopulating HSCs for several weeks (17). HSC differentiation during expansion increases HSC senescence and cell death process (18), but direct contact between stem cells and cellular microenvironment has been shown to play an essential role in self-renewal versus differentiation (19).
In this study, we observed that the expansion of HSCs in the cytokine supplemented culture compared to the co-culture system without cytokine have the highest apoptosis rate. Goldenberg et al.